key: cord-0716159-e3rxr6oq
authors: Navarro, Ryan; Nair, Rajeev; Peda, Andrea; Aung, Meiji Soe; Ashwinie, GS; Gallagher, Christa A; Malik, Yashpal S; Kobayashi, Nobumichi; Ghosh, Souvik
title: Molecular characterization of canine parvovirus and canine enteric coronavirus in diarrheic dogs on the island of St. Kitts: First report from the Caribbean region
date: 2017-08-15
journal: Virus Res
DOI: 10.1016/j.virusres.2017.08.008
sha: 33d10176a7a2bf866cb951955ddb3c4960424466
doc_id: 716159
cord_uid: e3rxr6oq

Although canine parvovirus (CPV) and canine enteric coronavirus (CCoV) are important enteric pathogens of dogs and have been studied extensively in different parts of the world, there are no reports on these viruses from the Caribbean region. During 2015–2016, a total of 104 diarrheic fecal samples were collected from puppies and adult dogs, with or without hemorrhagic gastroenteritis, on the Caribbean island of St. Kitts (KNA). By PCR, 25 (24%, n = 104) samples tested positive for CPV. Based on analysis of the complete deduced VP2 amino acid sequences, 20 of the KNA CPV strains were assigned to new CPV-2a (also designated as CPV-2a-297A). On the other hand, the VP2 genes of the remaining 5 strains were partially characterized, or could not be sequenced. New CPV-2a was the predominant CPV variant in St. Kitts, contrasting the molecular epidemiology of CPV variants reported in most studies from nearby North and South American countries. By RT-PCR, CCoVs were detected in 5 samples (4.8%, n = 104). Based on analysis of partial M-protein gene, the KNA CCoV strains were assigned to CCoV-I genotype, and were closely related to CCoV-I strains from Brazil. To our knowledge, this is the first report on detection and genetic diversity of CPV and CCoV in dogs from the Caribbean region, and underscores the importance of similar studies in the other Caribbean islands.

Viruses are important etiological agents of diarrhea in domestic and wild canids. Among them, canine parvovirus (CPV), a member of the family Parvoviridae, is a major cause of hemorrhagic gastroenteritis in dogs (Decaro and Buonavoglia, 2012; Miranda and Thompson, 2016) . CPV are small, nonenveloped viruses consisting of a single-stranded, negative sense DNA (∼5.2 kb) molecule (Berns and Parrish, 2013; Parrish, 1999; Reed et al., 1988) . The CPV genome contains two large open reading frames (ORF). The right ORF encodes 2 structural proteins (VP1 and VP2) by alternative splicing of the same mRNAs, whilst the left ORF codes for 2 nonstructural proteins (NS1 and NS2). The CPV VP2 capsid protein is antigenically significant, and has been implicated in governing host range restriction, tropism, and viral-host interactions (Decaro and Buonavoglia, 2012; Miranda and Thompson, 2016; Parrish, 1999) .

Most studies on molecular epidemiology of CPV are based on the VP2-encoding gene (Decaro and Buonavoglia, 2012; Miranda and Thompson, 2016; Truyen, 2006) . CPV emerged as a new enteric pathogen of domestic dogs in the late 1970s, possibly through host switching events involving a feline panleukopenia parvovirus, or a closely related virus (Berns and Parrish, 2013; Parrish, 1999) . As a result of accumulation of mutations in the VP2-encoding gene, the original CPV strain (strain CPV-2) eventually got replaced with antigenic variants CPV-2a, CPV-2b, CPV-2c, new CPV-2a, and new CPV-2b that are variously distributed in dog populations worldwide (Decaro and Buonavoglia, 2012; Miranda and Thompson, 2016) .

Although the current CPV vaccines, derived from the original CPV-2 strains, or CPV-2b strains, have been shown to confer protective immunity against CPV disease, and post-vaccination reactions have rarely been encountered in immunized dogs, the emergence of new genetic and antigenic variants underscores the importance of constant http://dx.doi.org/10.1016/j.virusres.2017.08.008 Received 16 May 2017; Received in revised form 10 July 2017; Accepted 21 August 2017 monitoring of evolution patterns of CPV strains circulating in dogs throughout the world (Decaro and Buonavoglia, 2012; Miranda and Thompson, 2016) .

Canine coronavirus (CCoV) (family Coronaviridae, genus Alphacoronavirus, species Alphacoronavirus-I) usually cause mild, selflimiting enteritis in dogs, although fatal disease has been observed with a pantropic variant of CCoV Buonavoglia, 2008, 2011; Decaro et al., 2013; Pinto et al., 2014) . CCoV are enveloped viruses with a single-stranded, positive sense RNA (27-31 kb) genome . The CCoV membrane (M) protein is the most abundant structural protein and has been shown to elicit antibodies, whilst the spike (S) glycoprotein is the main inducer of virus-neutralizing antibodies.

Based on analysis of the M-and/or S-protein encoding genes, CCoV strains have been classified into at least two genotypes, CCoV-I and CCoV-II Buonavoglia, 2008, 2011) . Recently, CCoV-II strains were further classified into two subtypes, CCoV-IIa (classical strains) and CCoV-IIb (strains arising from putative recombination events between CCoV-II and transmissible gastroenteritis virus of swine) Buonavoglia, 2008, 2011; Le Poder, 2011) . CCoVs have been detected in canine populations worldwide Buonavoglia, 2008, 2011) .

The Caribbean region has a sizeable dog population, and dogs with diarrhea, including those with hemorrhagic gastroenteritis are routinely presented at veterinary clinics on these islands. Although the prevalence and genetic diversity of CPV and CCoV in dogs have been extensively studied in different parts of the world including nearby Latin American countries, there are no reports on these important canine viruses from the Caribbean region so far. We report here the detection and molecular characterization of CPV and CCoV strains in dogs with diarrhea on the Caribbean island of St. Kitts (KNA).

During 2015-2016, a total of 104 diarrheic fecal samples were collected from puppies and adult dogs, with or without hemorrhagic gastroenteritis, at two veterinary clinics (the Ross University School of Veterinary Medicine Clinic, and the Ponds Veterinary Clinic) on the island of St. Kitts, Caribbean region. The samples were stored at −20°C until further analysis. The present study was conducted in compliance with good laboratory practice (GLP).

For PCR, viral DNA was extracted from the fecal samples using the QIAamp Fast DNA Stool Mini Kit (Qiagen Sciences, MD, USA). Samples were screened for the presence of CPV using a PCR-based detection assay targeting a 583-bp stretch of the 3′-portion of the VP2-encoding gene, as described previously (Buonavoglia et al., 2001) . In order to determine the CPV variant, a 1799 bp fragment of CPV genome containing the complete ORF of VP2 gene was amplified using a newly designed primer VP2F (5′-ATG AGT GAT GGA GCA GTT CAA CC-3′, corresponding to nucleotide [nt] 2787-nt 2809 of reference strain CPVb), and primer 555rev (Buonavoglia et al., 2001) . PCRs were performed using Platinum™ Taq DNA Polymerase (Invitrogen, CA, USA) following manufacturer's instructions. PCR-grade water was used as the negative control.

Viral RNA was extracted from fecal samples using the QIAamp Viral RNA Mini Kit (Qiagen Sciences, MD, USA). For detection of CCoVs in fecal samples, RT-PCR based on a partial stretch (409 bp) of M proteinencoding gene was performed as reported previously (Pratelli et al., 1999) . RT-PCRs were carried out using SuperScript ® III RT (Invitrogen, CA, USA) and Platinum™ Taq DNA Polymerase (Invitrogen, CA, USA) following manufacturers' instructions. We used PCR-grade water as the negative control.

For nt sequencing, PCR products were purified using the QIAquick PCR Purification Kit (Qiagen Sciences, MD, USA) according to manufacturer's protocol. Nucleotide sequences were obtained using the ABI Prism Big Dye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems, CA, USA) on an ABI Prism 3130 Genetic Analyzer (Applied Biosystems, CA, USA.). The PCR products were sequenced in both directions.

Homology search for related cognate sequences was performed using standard nt BLAST program (Basic Local Alignment Search Tool, www.ncbi.nlm.nih.gov/blastn). Multiple alignments of deduced amino acid (aa) sequences were performed using the CLUSTALW program (version ddbj, http://clustalw.ddbj.nig.ac.jp/) with default parameters. Phylogenetic trees were constructed using the MEGA (v5.2.2) software.

The GenBank accession numbers for nt sequences of complete, or partial ORF of VP2 genes of the KNA CPV strains, and partial M-protein encoding genes of the KNA CCoV strains are shown in Table 1 .

The federation of St. Kitts and Nevis is a twin island nation in the Lesser Antilles of the Caribbean region with a total human population of ∼55,000 (Map is shown in Supplementary Fig. S1 ). Although there are no official estimates on the canine population of St. Kitts, different breeds of domestic dogs, including a local island breed are kept as pets in many households on the island. Diarrhea, including hemorrhagic gastroenteritis is prevalent in domestic dogs on St. Kitts, as evident from clinical cases that are presented now and then at the two major veterinary clinics (the Ross University School of Veterinary Medicine Clinic [RVC] , and the Ponds Veterinary Clinic [PVC]) on the island. In the present study, CPV, or CCoV were detected in 30 (28.8%) of the 104 fecal samples obtained from diarrheic dogs at the two veterinary clinics on St. Kitts (Table 1) .

By PCR of the partial VP2 gene, a total of 25 (24%, n = 104) dogs tested positive for CPV. Among them, 15 dogs were presented with hemorrhagic gastroenteritis, whilst the remaining 10 dogs had severe diarrhea. All the CPV positive dogs were sporadic cases from different households across the island of St. Kitts. The age of the dogs that tested positive for CPV ranged from 3 days up to 3 years of age (Table 1) . Eighteen of the 25 CPV positive dogs were aged ≤ 6 months, corroborating previous observations that dogs up to 6 months may exhibit a greater risk of infection (Decaro and Buonavoglia, 2012; Miranda et al., 2015) . Most of the CPV positive samples were from mixed breeds (Table 1) , contradicting a previous observation that purebreds were more susceptible to CPV disease than mixed breeds (Kalli et al., 2010) . However, a few other studies have shown that breed may not be a risk factor (Miranda et al., 2015) .

Vaccination is crucial to control and prevent CPV disease (Decaro and Buonavoglia, 2012; Miranda and Thompson, 2016) . Lack of vaccination, incomplete vaccination schedules, or vaccine failures, primarily due to interference with maternal antibodies may predispose dogs to natural infection. Moreover, some studies have raised questions on the complete efficacy of the current CPV vaccines against the CPV antigenic variants, as these vaccines were derived from old CPV-2 strains (Decaro et al., , 2009 Decaro and Buonavoglia, 2012; Miranda and Thompson, 2016; Truyen, 2006) . In the present study, nearly half (11/25) of the CPV positive dogs did not receive any vaccine, whilst 2 puppies did not attain the recommended minimum age of vaccination (6 weeks) at the time of sampling (Table 1) . Among the vaccinated dogs that tested positive for CPV (4/25), only one animal had received two doses of the vaccine (Table 1) . Therefore, lack of, or inadequate protective immunity might have predisposed these dogs to CPV disease, although the role/s of other risk factors, such as endoparasitism and unsanitary environments cannot be ruled out (Miranda et al., 2015) . On the other hand, no information was available on the immunization status of the remaining CPV positive dogs (Table 1) . Information on vaccination status was available for only 26 of the 79 dogs that tested negative for CPV. Eleven of these dogs received one, or more doses of CPV vaccine before sampling, whilst 15 were not immunized against CPV.

To study the genotype nature and evolution of CPV strains circulating in St. Kitts, we determined the complete ORF nt sequences of VP2 genes of 20 KNA strains (Table 1 ). The deduced VP2 aa sequences of all the 20 KNA CPV strains exhibited 297-A, 426-N and 555-V, and therefore, were classified as new CPV-2a (CPV-2a-297A) strains (Table 2 ; Supplementary Fig. S2 ). On the other hand, the VP2 genes of the remaining 5 KNA strains were partially characterized, or could not be sequenced due to insufficient volumes of fecal samples (Table 1) . Other relevant mutations that may influence viral antigenicity and/or host range have also been reported in many of the recent CPV-2a/2b strains, such as G300D, Y324I and T440A (Decaro and Buonavoglia, 2012; Miranda and Thompson, 2016) . However, these mutations were not observed in the putative VP2 proteins of the KNA new CPV-2a strains (Table 2 , Supplementary Fig. S2 ).

The VP2 of KNA CPV strains shared 99-100% nt and deduced aa identities among themselves, and with those of other new CPV-2a strains. Nucleotide and deduced aa identities of 99% were also observed with the VP2 of the CPV-2, CPV-2a, CPV-2b, new CPV-2b, and CPV-2c strains. By phylogenetic analysis of complete ORF nt sequences of VP2 genes, the KNA strains clustered together, and were found to be more closely related to new CPV-2a strains than other CPV-2 variants (Fig. 1) , corroborating the observations at deduced aa level.

Although the present study is the first report on CPV variants from the Caribbean region, the molecular epidemiology of CPV has been documented in nearby North and South American countries (Miranda and Thompson, 2016) . In Ecuador and USA, CPV-2b and CPV-2c were more prevalent than CPV-2a. Only CPV-2c strains have been reported from Mexico and Paraguay. CPV-2c was the major variant in Argentina and Brazil (Calderón et al., 2012; Pinto et al., 2012) . CPV-2b was the major genotype in Canada (Gagnon et al., 2016) . In Uruguay, CPV-2c was the predominant variant during 2006-2010 (Miranda and Thompson, 2016) . However, new CPV-2a variants emerged in 2010 (Pérez et al., 2012) , and went on to become the major CPV-2 variant in Uruguay in 2011 (Maya et al., 2013) . A recent study reported CPV-2a as the major variant in Columbia (Duque-García et al., 2017) . In the present study, new CPV-2a was found to be the predominant CPV variant in domestic dogs on the Caribbean island of St. Kitts, contrasting the molecular epidemiology of CPV reported in most studies from North and South American countries.

By RT-PCR targeting a partial fragment of the M protein encoding gene, 5 of the 104 samples tested positive for CCoVs (Table 1) . Among these sporadic cases, one sample was from an adult dog with mild enteritis, whilst the remaining fecals were from puppies exhibiting mild to severe diarrhea. In the present study, high quality nt sequences of the partial M-protein gene (369 bp, excluding the 5′-and 3′-end primers) could be obtained for 4 of the KNA CCoV strains (Table 1) .

The partial M-protein gene of the KNA CCoV strains shared 100% nt sequence identities between themselves. With other CCoVs, the partial M protein gene sequences of the KNA CCoV strains shared maximum nt and deduced aa sequence identities of 99% and 100%, respectively, with those of several Brazilian CCoV-I strains. Absolute deduced aa sequence identities were also observed with cognate stretch of reference CCoV-I strain 23/03/ITA. On the other hand, nt and deduced aa identities of < 91% and < 94%, respectively, were observed with those of CCoV-II (CCoV-IIa and −IIb) strains. Alignment of partial deduced M protein aa sequences of the KNA CCoV strains with those of CCoV-I, and CCoV-IIa and −IIb strains is shown in supplementary Fig. S3 . By phylogenetic analysis of partial M protein genes, the KNA CCoV strains were found to be closely related to CCoV-I strains from Brazil within the CCoV-I cluster, and were distantly related to CCoV-II and feline coronavirus strains (Fig. 2) . Based on analysis of partial M-protein gene, the KNA CCoV strains were found to belong to CCoV-I genotype.

Whilst the molecular epidemiology of CCoV has been studied in different Asian and European countries (Cavalli et al., 2014; Buonavoglia, 2008, 2011; Decaro et al., 2010 Decaro et al., , 2013 Erles and Brownlie, 2009; Jeoung et al., 2014; McElligott et al., 2011; Ntafis et al., 2013; Soma et al., 2011; Wang et al., 2016) , there is a dearth of data on distribution of CCoV genotypes from the North and South Americas. In Brazil, CCoV-I and CCoV-II strains, including pantropic CCoV-IIa variants were found to be circulating in the domestic dog population (Costa et al., 2014; Pinto et al., 2014) . CCoV-IIa and −IIb genotypes have been associated with fatal enteritis in puppies in USA (Licitra et al., 2014) . Although CCoVs were detected at lower frequencies in St. Kitts, this is the first report on detection and genotyping Table 2 Comparison of evolutionary relevant amino acid (aa) residues of putative VP2 proteins of canine parvovirus (CPV) strains detected on the island of St. Kitts (KNA), Caribbean region, with those of CPV-2, CPV-2a, CPV-2b, CPV-2c, new CPV-2a, new CPV-2b, and vaccine strains. The KNA CPV strains are underlined. A dot '.' indicates an identical amino acid residue at cognate position of deduced VP2 aa sequence of the concerned CPV strain with that of reference strain CPV-b/USA/1978. Alignment of complete deduced aa sequences of putative VP2 proteins of the CPV strains is shown in supplementary Fig. S2 . Amino acid position a  80 87 93 101 103 232 267 297 b 300 305 321 323 324 375 426 c 440 555 560 564 568 570 Variant Strain 1978. b The S297A mutation has been found to be fixed in VP2 of recent CPV-2a and CPV-2b strains, and these variants are sometimes designated as new CPV-2a and new CPV-2b strains (Decaro and Buonavoglia, 2012; Miranda and Thompson, 2016) . In a recent study, new CPV-2a and new CPV-2b strains have also been designated as CPV-2a-297A and CPV-2b-297A, respectively (Zhou et al., 2017) . c Amino Acid residue 426 of VP2 constitutes the sole basis of differentiating the new CPV-2a (426-N), new CPV-2b (426-D), and CPV-2c (426-E) strains (Decaro and Buonavoglia, 2012; Miranda and Thompson, 2016) . d New CPV-2a strains exhibiting G300D are also referred to as CPV-2a-297A300D (Zhou et al., 2017) . 

We reported here the detection and molecular characterization of CPV and CCoV on the Caribbean island of St. Kitts. Although PCR/RT-PCR have been used extensively for detection of CPV/CCoV in many studies including recent reports, these screening assays have been shown to be relatively less sensitive than qPCR/RT-qPCR (Decaro et al., 2005a,b; Duque-García et al., 2017; Kumar and Nandi, 2010; Miranda and Thompson, 2016; Ntafis et al., 2013; Wang et al., 2016) . One of the limitation of this study was the use of PCR/RT-PCR as screening assays. However, considering the lack of data from the Caribbean region, the present study primarily focused on detection and molecular characterization of circulating strains rather than strictly monitoring prevalence. New CPV-2a (CPV-2a-297A) was found to be the predominant CPV variant on St. Kitts. CPV-2a has been shown to be the major variant in Asia, whilst CPV-2c, or CPV-2b were predominant in most studies from the North and South Americas (Miranda and Thompson, 2016; Zhou et al., 2017) . However, the predominance of CPV-2a variants in recent studies from Columbia, Uruguay, and St. Kitts pointed towards the changing epidemiology of CPV in this part of the world. The KNA CCoV strains were genetically closely related to Brazilian CCoV-I strains, which may be attributed to the geographical proximity of the Caribbean region to Latin American countries. Although this is the first report on detection and genetic diversity of CPV and CCoVs from the Caribbean region, the present study was based on a single island. In order to gain vital insights into the molecular epidemiology of these important canine viruses across the entire Caribbean region, similar studies are required in the other islands. Fig. 2 . Phylogenetic tree constructed by the Maximum Likelihood (ML) method from nucleotide sequences of partial M-protein encoding genes (369 bp) of the KNA CCoV strains with those of CCoV-I, CCoV-II, and feline coronavirus (FCoV) strains. The tree was statistically supported by bootstrapping with 1000 replicates, and phylogenetic distances were measured using the Tamura-3parameter model of substitution. The clustering patterns of KNA CCoV strains were validated by creating ML trees with other mathematical models, such as the Jukes-Cantor model, Kimura 2-parameter model, and Hasegawa-Kishino-Yano model (data not shown). In the tree, the positions of the KNA CCoV strains are shown by dark circles. GenBank accession numbers are shown in parentheses. Bootstrap values < 70% are not shown. Scale bar, 0.05 substitutions per nucleotide.

Parvoviridae

Evidence for evolution of canine parvovirus type 2 in Italy

Study of canine parvovirus evolution: comparative analysis of fulllength VP2 gene sequences from Argentina and international field strains

Detection and genetic characterization of Canine parvovirus and Canine coronavirus strains circulating in district of Tirana in Albania

Molecular characterization of canine coronavirus strains circulating in Brazil

An update on canine coronaviruses: viral evolution and pathobiology

Canine Coronavirus: not only an enteric pathogen

Canine parvovirus-A review of epidemiological and diagnostic aspects, with emphasis on type 2c

A real-time PCR assay for rapid detection and quantitation of canine parvovirus type 2 in the feces of dogs

Genotype-specific fluorogenic RT-PCR assays for the detection and quantitation of canine coronavirus type I and type II RNA in faecal samples of dogs

Evidence for immunisation failure in vaccinated adult dogs infected with canine parvovirus type 2c

Severe parvovirus in a 12-year-old dog that had been repeatedly vaccinated

Recombinant canine coronaviruses in dogs

Western European epidemiological survey for parvovirus and coronavirus infections in dogs

European surveillance for pantropic canine coronavirus

Prevalence and molecular epidemiology of Canine parvovirus 2 in diarrheic dogs in Colombia, South America: a possible new CPV-2a is emerging?

Sequence analysis of divergent canine coronavirus strains present in a UK dog population

Canine parvovirus type 2b is the most prevalent genomic variant strain found in parvovirus antigen positive diarrheic dog feces samples across Canada

M gene analysis of canine coronavirus strains detected in Korea

Factors affecting the occurrence, duration of hospitalization and final outcome in canine parvovirus infection

Development of a SYBR Green based real-time PCR assay for detection and quantitation of canine parvovirus in faecal samples

Feline and canine coronaviruses: common genetic and pathobiological Features

Genotypic characterization of canine coronaviruses associated with fatal canine neonatal enteritis in the United States

Phylodynamics analysis of canine parvovirus in Uruguay: evidence of two successive invasions by different variants

Detection and genetic characterization of canine parvoviruses and coronaviruses in Southern Ireland

Canine parvovirus: the worldwide occurrence of antigenic variants

Factors affecting the occurrence of canine parvovirus in dogs

Canine coronavirus, Greece. Molecular analysis and genetic diversity characterization

Recent spreading of a divergent canine parvovirus type 2a (CPV-2a) strain in a CPV-2c homogenous population

Host range relationships and the evolution of canine parvovirus

Typing of canine parvovirus strains circulating in Brazil between

Characterization of pantropic canine coronavirus from Brazil

Development of a nested PCR assay for the detection of canine coronavirus

Nucleotide sequence and genome organization of canine parvovirus

Detection and genotyping of canine coronavirus RNA in diarrheic dogs in Japan

Evolution of canine parvovirus − a need for new vaccines?

Co-circulation of canine coronavirus I and IIa/b with high prevalence and genetic diversity in Heilongjiang Province

The genetic evolution of canine parvovirus − A new perspective

We are thankful to Dr 

Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.virusres.2017.08.008.